Electrophoresis involves the separation of mixtures by differential migration of components through a transport medium or support in an electric field. Many molecules and particles in aqueous solution acquire an electrical charge due to ionization and thus move in response to an external electric field. The charged particles may be simple ions, complex macromolecules, viruses, colloids or even living cells. The rate of their migration depends generally upon the amount of charge, the size and shape of the particle, and the properties of the solvent and support.
Electrophoresis in a gel support is an important method of separating proteins, nucleic acids and other such macromolecules in mixture. When an electric field is applied to the support at a given pH, the macromolecules migrate toward the oppositely charged electrode. When the support does not exert any influence, the higher the ratio of charge to mass, the faster the molecules migrate, and, application of current across the support results in a series of bands according to mass/charge ratio.
For molecules such as DNA, or proteins of analogous amino acid composition treated with a detergent such as SDS, the mass/charge ratio is often virtually identical for all these components of a mixture, since the DNA or protein molecules acquire a fairly uniform charge per repeating subunit which is roughly the same. When electrophoresed on a support having a pore size smaller than the molecules' cross section, the migration rate depends inversely on size. The mixture of macromolecules is thereby eventually separated into a series of distinct bands dependent mostly on their relative size.
The electrophoresis is generally terminated when the leading band has migrated through most of the available gel. The bands can be identified by suitable means such as staining, optical scanning and the like procedures, and the macromolecules can be recovered by cutting out and solubilizing the corresponding portions of the gel. This can be done, for example, by electroelution from the gel or by chemical or physical disruption of the gel structure followed by appropriate purification techniques.
A variety of materials can be used to obtain the gels. The gels must provide a matrix wherein the pore size is smaller than the cross-sectional dimension of the molecules to be separated, or mass/charge consideration will be determinative, and little separation will be achieved when mass/charge ratios are nearly the same. Thus, in free solution, or in gels of very large pore size, all linear DNAs have the same mobility regardless of size. It is only because of the sieving effects of the gel, wherein larger DNA molecules must find more circuitous paths, thereby slowing their progress, that separation can be achieved. But for very small pore sizes, very large molecules tend hardly to move at all, and the larger the molecules desired to be separated, the larger the pore size must be. Agarose, which is a naturally occurring linear polysaccharide of galactose and 3,6-anhydrogalactose, is particularly useful as the electrophoretic support medium since it permits the separation of large molecules such as viruses, enzyme complexes, lipoproteins and nucleic acids which are sometimes outside the useful pore size with polyacrylamide gel electrophoresis. A large variety of agaroses and modified agaroses are available commercially. They are usually used in concentrations ranging from about 0.1 to about 2.5% by weight, which gives a pore size of 10-100 .ANG.. Polyacrylamide gels are also commonly used for smaller molecules of interest as they have smaller pore sizes, of the order of 10 .ANG. or less. A variety of support materials has been used, and the invention is not limited to any particular support medium. However, agarose and polyacrylamide are clearly the most common and best studied, and therefore preferred by most practitioners.
Notwithstanding the foregoing, the use of conventional agarose or polyacrylamide gel electrophoresis has not generally been ideally suited for separation of the largest deoxyribonucleic acid (DNA) molecules, that is, molecules which are larger than about 2.times.10.sup.5 base pairs (bp) or about 200 kb. Most practical work has been confined to molecules less than about 2.times.10.sup.4 bp or about 20 kb. Although typical DNA molecules employed in genetic engineering applications are within this lower size range, the DNA molecules in chromosomes are larger.
Further background information on conventional gel electrophoresis of DNA can be had by reference to a text such as Rickwood and Hames, Gel Electrophoresis of Nucleic Acids: A Practical Approach, IRL Press, Oxford, UK, particularly chapter 2, "Gel Electrophoresis of DNA", by Sealey and Southern.
For background information on attempts to achieve separation of very large DNA molecules by conventional gel electrophoresis, reference can be had to papers by Fangman, Nucleic Acids Res. 5: 653-665 (1978); and Serwer, Biochemistry 19, 3001-3004 (1980). Both reports relate to the use of dilute gels, since here the pore sizes will be more in keeping with the size of the molecules whose separation is desired. In the former paper, using very dilute agarose gels (which are difficult to handle) and low voltages (which require long running times), Fangman was able to achieve a mobility ratio of bacteriophage G DNA (approximately 750 kb, where 1 kb=1 kilobase pair=1000 base pairs) to bacteriophage T4 DNA (approximately 170 kb) of approximately 1.4. Molecules larger than bacteriophage G were not investigated. So also in the latter paper, Serwer found that the best conditions involved dilute agarose gels run at low voltages. Molecules larger than approximately 170 kb were not investigated.
Recently, a modified gel electrophoresis technique for separating large DNA molecules was disclosed by Schwartz et al., Cold Spring Harbor Symp. Quant. Biol. 47: 189-195 (1983); Schwartz and Cantor, Cell 37: 67-75 (1984); Smith and Cantor, Nature (1986) 319: 701-702; and Cantor and Schwartz, U.S. Pat. No. 4,473,452. According to their disclosed technique, the DNA molecules are separated by subjecting the gel medium alternately to two non-uniform electric fields having co-planar directions which are transverse to each other. The two fields alternate between respective high and low intensities out of phase with each other at a frequency related to the mass of the particles. Because the fields are transversely applied, the DNA molecules migrate in a direction that lies between the two field directions.
Although the disclosed Cantor and Schwartz technique has been applied with success to separate DNA molecules present in the chromosomes of lower organisms such as yeast and protozoans, the bands are somewhat distorted and nonparallel, presumably because there is asserted to be an advantage, in their approach, of using non-uniform fields. It is thus difficult to make lane-to-lane comparisons between samples as is obtained in conventional gel electrophoresis. Moreover, the transverse-field gel electrophoresis technique requires complex electrode geometries. Although the theoretical minimum is three, no devices have been described that contain fewer than four, and it is common for devices to feature whole arrays of electrodes. Furthermore, the precise positioning of the electrodes has dramatic effects on the results obtained. Consequently, transverse-field-alternation gel electrophoresis does not provide for convenient gel electrophoresis practice.
Implementation of the transverse-field technique (also defined as orthogonal-field-alternation gel electrophoresis, or OFAGE) and applications to the chromosomal DNA molecules from yeast are described by Carle and Olson, Nucleic Acids Res. 12: 5647-5664 (1984). A description of the complete analysis of the set of chromosomal DNA molecules from yeast using the transverse-field technique is further reported by Carle and Olson. Proc Natl Acad Sci (USA) 82: 3756-3760 (1985).
Other background information on the application of the transverse-field technique of gel electrophoresis to chromosomal DNA molecules is provided by Van der Ploeg et al., Cell 37: 77-84 (1984); Van der Ploeg et al., Cell 39: 213-221 (1984); and Van der Ploeg et al., Science 229: 658-661 (1985).